The use of nucleic acid (DNA and RNA) as antisense or antigene drugs, to bind to DNA or RNA of disease-causing proteins to prevent their production, has been hampered by features of the DNA and RNA. For example, the negatively charged phosphodiester linkages of double- and triple-stranded DNA and RNA reside side by side, causing considerable charge-charge electrostatic repulsion, particularly at low (physiological) ionic strength. This feature, as well as the susceptibility of DNA and RNA to damage from nuclease activity, limits the usefulness of RNA and DNA as therapeutic agents. (See, Temsamami and Guinot, Biotechnol. Appl. Biochem. 26:65 (1997); Uhlmann and Peyman, Chem. Rev. 90:543 (1990); Crooke, Anticancer Drug Des. 6:609 (1991); and Crooke, Ann. Rev. Pharmacol. Toxicol. 32:329 (1992)).
Among other requirements, successful development of antisense therapeutics presupposes the oligonucleotides a) be stable in vivo, b) have improved permeability and cellular uptake and c) have greater binding affinity with high specificity. (Miller and Ts'o, Anticancer Drug Des. 2:117 (1987); Milligan et al., J. Med. Chem. 36:1923 (1993); and Mesmaeker et al., Curr. Opin. Struct. Biol. 343 (1995)).
Suggestions have been made to replace the phosphodiester linkages by other linkages that are either neutral or positively charged and resistant toward nuclease degradation in order to provide more effective antigene/antisense agents. (See, Crooke, (1992), supra, Cook, In Antisense Research and Applications, Lebleu (Ed.), CRC Press, Boca Raton, Fla., (1993), p. 149; and Morvan et al., J. Med. Chem. 36:280 (1993)). In addition, modification of oligonucleotides so as to enhance cellular uptake has been considered. (Cook et al., supra).
Antisense oligonucleotides having various backbone modifications have been prepared (see Bennett, Biochemical Pharmacology 55:9 (1998); Alama et al., Pharmacological Research 36:171 (1997); Manoharan, Designer Antisense oligonucleotides: conjugation chemistry and functionality placement, CRC Press, Boca Raton, Fla. (1993); and Mesmaeker et al., Pure Appl. Chem. 69:437 (1997)).
The replacement of the phosphate linkages in DNA and RNA by achiral guanido groups providing a new class of guanidinium (g) linked nucleosides which are designated as DNG has been reported. (Dempcy et al., Proc. Natl. Acad. Sci. USA 91:7864 (1994); Dempcy et al., Proc. Natl. Acad. Sci. USA 92 (1995); Dempcy et al., J. Am. Chem. Soc. 117:6140 (1995); and Dempcy et al., Proc. Natl. Acad. Sci. USA 93:4326 (1996)).
Some examples of backbone-modified oligonucleotides having different electrostatic atttractions include: peptide (PNA-neutral) (Egholm et al., J. Am. Chem. Soc. 114:1895 (1992); and Nielsen and Haaima, Chem. Soc. Rev. 73 (1997)); PHONA (Peyman et al., Angew. Chem., Int. Ed. Engl. 35:2636 (1996)); methyl phosphonate (DNAmp-neutral) (Stein and Cheng, Science 261:1004 (1993); and Tseng and Ts'o, Antisense Res. Dev. 5:251 (1995)); phosphorothioate (DNAs-anionic) (Cook, supra; Morvan et al., supra and Marshall and Caruthers, Science 259:1564 (1993)); phosphoramidate (Gryaznov and Chen, J. Am. Chem. Soc. 116:3143 (1994); amido (Mesmaeker et al., Angew, Chem. Int. Ed. Engl. 35:2790 (1996); MMI (Sanghvi et al., Nucleosides Nucleotides 16:907 (1997); boronated oligonucleotides (Sood et al., J. Am. Chem. Soc. 111:9234 (1989); Sood et al., J. Am. Chem. Soc. 223:9000 (1990) and Spielvogel et al., Pure Appl. Chem. 63:415 (1991)); ethylmorpholino and dimnethylamino phosphoramidates (Jung et al., Nucleosides and Nucleotides 13:1597 (1994) and Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988)); aminomethyl phosphonates (Huang et al., Bioconjugate Chem. 5:47 (1994); and guanido (Vasseur et al., J. Am. Chem. Soc. 114:4006 (1992); Blattler et al., J. Am. Chem. Soc. 120:2674 (1991); James et al., Nucleosides Nucleotides 16:1821 (1997); Jones et al., J. Org Chem. 58:2983 (1993); Mesmaeker et al., Acc. Chem. Res. 28:366 (1995); Rao et al., Nucleosides Nucleotides 13:255 (1997); Stirchak and Summerton, J. Org. Chem. 52:4202 (1987); and Thibon et al., J. Org. Chem. 62:4635 (1997)) (See FIG. 1).
Small positively charged oligonucleotides (DNG) show unprecedented binding to nucleic acids with retention of specificity. (Dempcy et al., Proc. Natl. Acad. Sci. USA 92 (1995), supra and Dempcy et al., J. Am. Chem. Soc. 117:6140 (1995), supra; and Browne et al., Proc. Natl. Acad. Sci. USA 92:7051-7055 (1995)). The nonionic oligonucleotide DNAmp exhibits the ability to be transported into cells by passive diffusion/fluid phase endocytosis and is more resistant to degradation than DNA. (Cook et al., supra). Both DNAmp and DNAs, however have individual limited drawbacks of stereoisomeric complexity (Huang et al., supra), solubility (DNAmp) and toxicity (DNAs). (Morvan et al., supra, and Agrawal et al., Nucleosides Nucleotides 16:927-936 (1997)). These findings have led recently to the development of mixed backbone oligonucleotides(MBOs) where the phosphorothioates and methyl phosphonates have been alternated in an oligonucleotide backbone to produce improved antisense properties. (Morvan et al., supra, Agrawal et al., supra and Iyer et al., Tetrahedron 52:14419-14436 (1996)).
There remains a need for oligonucleotides that may perform better as antisense or antigene drugs, for example by having stronger affinity for DNA and RNA, as a result of changes in charge characteristics and resistance to nuclease degradation which can form stable constructs.